PROJECT SUMMARY The central goal of this proposal is to develop and provide proof-of-concept testing for an in situ genome editing platform to accomplish programmed gene modification (including sequence deletion and sequence insertion) in endogenous genes of hematopoietic stem and progenitor cells (HSPCs). If successful, our studies could provide an alternative to current transplantation-based paradigms being pursued in both academia and industry to achieve curative therapy for genetic blood diseases, including Sickle Cell Disease (SCD) and ?- thalassemia. While such strategies, in which allogeneic or ex vivo modified autologous HSPCs are used to reconstitute the patient's blood forming system with hematopoietic progenitors that produce functional, non-sickling ?-globin, represent a reasonable and even promising approach for rescuing defective hemoglobin production, they carry inherent risks from transplantation-associated toxicities, and have suffered from the ongoing challenge of achieving sufficient engraftment to support therapeutic hematopoietic reconstitution in animal models and patients. For these reasons, we choose here to pursue a radically different approach. The overall hypothesis driving this work is that by using in vivo functional gene modification strategies we can develop a broadly applicable approach to correct the phenotypic and functional defects of erythrocytes in both SCD and ?-thalassemia. Our experimental approach will build on our extensive preliminary data and novel resources documenting the in vivo transduction of HSPCs by adeno-associated viruses (AAVs) carrying genome modifying nucleases and adapt this system to enable sequence-specific homology directed recombination (HDR) at the ?-globin locus. We will then test the therapeutic utility of this approach using disease-relevant SCD model mice. Success in these pre-clinical studies will provide critical proof-of-concept data to support follow on studies to investigate this approach as a different, and possibly safer and more accessible, clinical path for achieving therapeutic gene editing in SCD, ?-thalassemia and possibly other hematologic disorders. In addition, this work may open new avenues for research, enabling future applications of this technology for the experimental interrogation of gene function in endogenous HSPCs, something that currently requires the generation of new transgenic mice or use of transplant systems that irrevocably alter the host environment and lead to oligoclonal hematopoiesis that is not normally seen in the blood system in young, healthy animals. Our proposed studies thus hold promise for both basic and translational impact.